Development Of Ex Vivo Expansion System For Mesenchymal Stem Cells By Using Spinner Flasks And Microcarriers

Mesenchymal stem cells(MSCs),which have differentiation potential,self-renewal capability and low immunogenicity,are most promising to be applied as regenerative medicine.Several therapeutic products based on MSCs have been translated into clinical applications.However,in general,therapeutical doses required for treatments are large,while the quantity of MSCs derived from tissue is extremely low.The traditional two-dimensional serum-containing culture system has been applied for ex vivo expansion of MSCs,which results in compromised quality,quantity,and safety of cell preparations.Therefore,in view of the high demand for generating clinical grade MSCs,in this study MSCs from rat bone marrow and adipose tissue(designated as bMSCs and aMSCs,respectively)were isolated,the effects of a long-term culture on MSCs were explored,a variety of microcarriers were explored for ex vivo expansion of MSCs,and eventually,a large array of components were evaluated on ex vivo culture of MSCs with the objective to developing a serum-free medium suitable for MSCs.Comprehensive characterizations of MSCs regarding cell morphology,proliferation,multi-lineage differentiation potential,colony-forming capability and immunophenotype were performed.First,bMSCs and aMSCs were isolated and comparative analyzed.No apparent differences could be noticed concerning the morphology between bMSCs and aMSCs.The long-term subculture made both types of cells smaller,weakened their colony forming ability,and stimulated the proliferation rate.However,bMSCs demonstrated better proliferation and colony forming ability than aMSCs,and only bMSCs could maintain their ability to differentiate after a long-term subculture.No significant difference was observed about the expression of some surface markers(i.e.,CD29+/CD90+/CD34-/CD45-)no matter cell types or population doublings,while the expression of CD44,CD73 and CD 106 was dependent on both cell type and subculture passage.Second,the expansion efficiency of MSCs varied with the type of microcarriers and the origin of MSCs.It was shown that bMSCs on Cytopore 2 and aMSCs on both Cytodex 1 and Cytodex 3 could maintained their proliferation,self-renewal capability and differentiation potential,and their actual maximum expansion folds were 4.56,3.53 and 1.79 respectively within 14 days of culture.On the other hand,although bMSCs on Cultispher S and aMSCs on both Cultispher S and Cytopore 2 could achieve 4.84,4.23 and 3.36 folds of expansion,respectively,bMSCs and aMSCs were unable to maintain their colony forming ability or differentiation ability.Finally,a large array of bioactive molecules including insulin,non-essential amino acid solution,L-VC,EGF,PDGF-BB and Collagen I,were demonstrated to be able to stimulate the proliferation of bMSCs when applied in different combinations.As a consequence,a serum-free medium,opt-2,which was suitable for the growth of bMSCs,while maintaining the adherence,proliferation,surface marker expression and differentiation,was successfully developed.Collectively,this study not only explored the effects of long-term culture on MSCs derived from different tissue origins,but also developed the optimal microcarrier and serum-free medium for in vitro expansion of MSCs.However,both the microcarrier culture system and the serum-free medium(i.e.,opt-2)need further optimization to increase the expansion efficiency for MSCs.The present work provides a profound basis for ex vivo expansion of clinical grade MSCs and thereby promoting expanded clinical applications of MSCs.